© 1997 American Heart Association, Inc.
Articles |
Peroxidation of LDL From Combined-Hyperlipidemic Male Smokers Supplied With ¯-3 Fatty Acids and Antioxidants
From the Institute for Nutrition Research, University of Oslo (I.R.B, C.A.D, B.S, K.S, B.H, M.S.N), Ullevål University Hospital (I.H, I.S, H.A), Petter Möller Orkla ASA (K.S), Oslo, Norway, and Allegheny University of the Health Sciences, MCP Hahnemann School of Medicine (S.L.K), Philadelphia, Pa.
Correspondence to Marit S. Nenseter, Institute for Nutrition Research, University of Oslo, Songsvannsvn. 9, 0450 Oslo, Norway. E-mail marit.nenseter{at}basalmed.uio.no
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Abstract |
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Abstract The effects of marine ¯-3 polyunsaturated fatty acids (FAs) and antioxidants on the oxidative modification of LDL were studied in a randomized, double-blind, placebo-controlled trial. Male smokers (n=41) with combined hyperlipidemia were allocated to one of four groups receiving supplementation with ¯-3 FAs (5 g eicosapentaenoic acid and docosahexaenoic acid per day), antioxidants (75 mg vitamin E, 150 mg vitamin C, 15 mg ß-carotene, and 30 mg coenzyme Q10 per day), both ¯-3 FAs and antioxidants, or control oils. LDL and human mononuclear cells were isolated from the patients at baseline and after 6 weeks of supplementation. LDL was subjected to cell-mediated oxidation by the patients' own mononuclear cells, as well as to Cu2+-catalyzed and 2,2'-azobis-(2-amidinopropane hydrochloride) (AAPH)–initiated oxidation. Extent of LDL modification was measured as lag time, the formation rate of conjugated dienes (CDs), the maximum amount of CDs formed, formation of lipid peroxides, and the relative electrophoretic mobility of LDL on agarose gels. Dietary supplementation with ¯-3 FAs increased the concentration of total ¯-3 FAs in LDL and reduced the concentration of vitamin E in serum. The ¯-3 FA–enriched LDL particles were not more susceptible to Cu2+-catalyzed, AAPH-initiated, or autologous cell–mediated oxidation than control LDL. In fact, enrichment with ¯-3 FAs significantly reduced the formation rate of CDs when LDL was subjected to AAPH-induced oxidation. Supplementation with moderate amounts of antioxidants significantly increased the concentration of vitamin E in serum and increased the resistance of LDL to undergo Cu2+-catalyzed oxidation, measured as increased lag time, reduced formation of lipid peroxides, and reduced relative electrophoretic mobility compared with control LDL. Supplementation with ¯-3 FAs/antioxidants showed oxidizability of LDL similar to that of control LDL and ¯-3 FA–enriched LDL. In conclusion, ¯-3 FAs neither rendered the LDL particles more susceptible to undergo in vitro oxidation nor influenced mononuclear cells' ability to oxidize autologous LDL, whereas moderate amounts of antioxidants protected LDL against oxidative modification.
Key Words: antioxidants • human mononuclear cells • hyperlipidemia • oxidized LDL • ¯-3 fatty acids
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Introduction |
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Epidemiological and experimental studies suggest that the marine ¯-3 FAs EPA and DHA have beneficial effects with regard to the development of cardiovascular diseases.1 2 3 4 However, the mechanisms by which dietary ¯-3 FAs exert their effects are not fully understood. Several lines of evidence suggest that oxidatively modified LDL plays an important role in the development of atherosclerosis.5 6 Oxidative modification of LDL involves peroxidation of polyunsaturated FAs. Dietary ¯-3 FAs are incorporated into lipoproteins, thereby potentially affecting the susceptibility of LDL to oxidative modification. There are conflicting results, however, between studies on the effects of ¯-3 FA supplementation on LDL oxidizability.7 8 9 10 11 12 13 14 15 16 17 In some of the studies, enhanced peroxidation of LDL was observed,7 8 9 10 11 whereas other studies12 13 14 15 16 17 showed no effect of dietary ¯-3 FAs on LDL oxidation. In animal studies, including those in rabbits, monkeys, and pigs, ¯-3 FA ingestion either increased or reduced the susceptibility of LDL to in vitro oxidation.18 19 20
During supplementation with fish oil, ¯-3 FAs are incorporated not only into lipoproteins but also into cellular membranes in the body. Monocytes, endothelial cells, and smooth muscle cells are able to oxidize LDL by cell-derived oxidants like H2O2 and superoxide radicals.21 Since ¯-3 FA supplementation may reduce free-radical production in stimulated human monocytes and polymorphonuclear cells,22 23 24 changes in cellular free-radical production due to ¯-3 FAs may in turn influence cell-mediated oxidative modification of LDL in vivo.
The important questions of whether supplementation with ¯-3 FAs renders the LDL particles more prone to oxidative modification and influences the cells' ability to oxidize LDL have not yet been conclusively answered. Furthermore, high concentrations of antioxidants have been shown to counteract enhanced the susceptibility of ¯-3 FA–enriched LDL to oxidative modification.7 9 10 Another important question, therefore, is whether moderate amounts of a mixture of various antioxidants can protect LDL against oxidation. To address these questions, we conducted a randomized, double-blind, placebo-controlled study of ¯-3 FA and antioxidant supplementation to male smokers with combined hyperlipidemia. In an attempt to better reflect events occurring in vivo, we isolated LDL as well as peripheral blood mononuclear cells from each patient. LDL was subjected to oxidation by metal ion–dependent and –independent methods as well as by the patients' own mononuclear cells.
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Methods |
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Materials
Gelatin capsules of ¯-3 FAs and control FAs as well as antioxidant control capsules were provided by Petter Möller Orkla ASA. Antioxidant capsules (Redoxon) were produced by Hoffman–La Roche England. Ham's F-10 and gentamicin were obtained from Bio Whittaker. PMA, BHT, standard FA methyl esters, and C17:0 were purchased from Sigma Chemical Co. Anti–glycophorin A Dynabeads M-450 were purchased from Dynal AS and AAPH from Polysciences Inc. Polymorphprep was delivered by Nycomed Pharma AS, and tissue-culture dishes were supplied by Costar. The BCA protein assay kit was obtained from Pierce Laboratories Inc. Kits for determination of total cholesterol, triacylglycerol, and phospholipids in LDL were purchased from Bio Merieux, and a kit for determination of lipid peroxides was from Kamiya Biomedical Co. Paragon lipoprotein electrophoresis agarose gels were supplied by Beckman Instruments, Inc, and Corning precast agarose gels were from Fisher Scientific. Vacutainer tubes for blood collection were delivered by Becton Dickinson.
Subjects
Forty-two male smokers with combined
hyperlipidemia were recruited from a continuous
screening program for risk factors among 40-year-old men and women in
Oslo, Norway. Inclusion criteria were age between 40 and 60 years,
smoking 
10 cigarettes/d, cholesterol levels between 6 and
9 mmol/L, and triacylglycerol levels
between 2 and 5 mmol/L. Patients with heart, kidney, liver,
or malignant diseases and vegetarians, alcoholics, or drug abusers were
excluded. All subjects were required to stop their intake of cod liver
oil, fish oil, and vitamin supplementation at least 3 months before the
start of the study. None of the patients took any prescribed drugs
during the study. All participants except one completed the study
without major problems. That patient did not complete the trial because
of a nonfatal myocardial infarction.
Study Design
The present study was double-blind, randomized, and placebo
controlled. The patients were randomly allocated to one of four groups
receiving supplementation with either ¯-3
polyunsaturated FAs (n=11), antioxidants (n=11), both
¯-3 FAs and antioxidants (n=11), or control oils (n=9).
The subjects received eight ¯-3 FA or FA control
capsules and one antioxidant or antioxidant control capsule daily for 6
weeks. Eight ¯-3 FA capsules provided 5 g of EPA
and DHA per day. The FA control capsules contained 8 g of oil with
an FA pattern similar to an ordinary Norwegian diet (Table 1
). The antioxidant capsule contained 75
mg vitamin E, 150 mg vitamin C, and 15 mg ß-carotene dissolved in 300
mg peanut oil, whereas the antioxidant control capsule contained peanut
oil only (Table 1). Coenzyme Q10 (30 mg/d) was
supplied in the ¯-3 FA or FA control capsules given to
the groups receiving antioxidants. Pill counts of unused capsules
indicated that overall compliance was satisfactory, with <2% of the
provided capsules returned. At baseline and after 6 weeks of
supplementation, all blood samples were drawn from the patients after
an overnight fast and 90 minutes after smoking 2 to 3 cigarettes.
Assessment of dietary intake was performed by a self-administered,
quantitative, food-frequency questionnaire25 26 27 at
baseline. Informed consent was obtained from all participants, and the
study protocol was approved by the Regional Committee of Medical Ethics
and by the Norwegian Health Authorities.
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Isolation of LDL
LDL was isolated from freshly prepared heparin-plasma
(Vacutainer tubes containing 143 USP units sodium heparin) collected
from the patients at baseline and after 6 weeks by sequential
ultracentrifugation in a Beckman L-80
ultracentrifuge. Plasma was adjusted to a density of 1.019
g/mL by adding solid NaBr. Centrifugation was
performed in a type 80 Ti fixed-angle rotor at 80 000 rpm for 5 hours
(1.3x108 g · minutes) at 10°C. After
centrifugation the top layer was removed. The
LDL-containing fraction was adjusted to a density of 1.063 g/mL
and centrifuged again at 80 000 rpm for 5 hours
(1.3x108 g · minutes) at 10°C. The top
layer containing LDL was collected and dialyzed extensively against PBS
(0.15 mol/L NaCl, 20 mmol/L sodium phosphate, pH
7.4). Protein concentrations were determined by BCA protein assay (see
"Materials"). The intra-assay coefficient of variation for protein
determination was 2.6% (n=10). Cholesterol,
triacylglycerol, and phospholipids in the LDL
fraction were determined enzymatically by kit methods (see
"Materials"). The intra-assay coefficient of variation was 3.9%,
3.8%, and 6.1% for measurement of cholesterol,
triacylglycerol, and phospholipid, respectively
(n=10). Prior to oxidation and cryopreservation, LDL was diluted with
PBS to obtain a final concentration of 1.5 mg LDL protein per
milliliter. One aliquot of LDL was used for cell-mediated oxidation
immediately.
Cryopreservation of LDL
An aliquot of LDL was cryopreserved in 10% sucrose (final
concentration).28 Sucrose (50% sucrose, 150
mmol/L NaCl, and 0.24 mmol/L EDTA, pH 7.4) was added
to freshly isolated LDL and the samples were frozen at -80°C. LDL
remained frozen for 6 to 8 months. LDL was thawed by immersion in a
37°C water bath. Prior to Cu2+-catalyzed and
AAPH-initiated oxidation, sucrose was removed by dialysis against PBS.
Rumsey et al28 reported no difference in the formation of
TBARS in Cu2+-oxidized LDL stored in 10% sucrose for at
last 18 months compared with freshly isolated LDL.
Isolation of Peripheral Blood Mononuclear
Cells
The cells were isolated from citrated, freshly collected blood
(Vacutainer tubes containing 0.129 mol/L trisodium citrate) from
patients at baseline and after 6 weeks of supplementation. The blood (5
mL) was immediately layered over 5 mL Polymorphprep. After
centrifugation at 480g (1600 rpm) for 30
minutes at 22°C in a swing-out rotor, two leukocyte bands were
visible. The top band, consisting of mononuclear cells, was removed and
washed with 15 mL Ham's F-10 (740g for 10 minutes) and
resuspended in 1 mL medium. Contaminating erythrocytes were removed by
addition of a 50-µL suspension of anti–glycophorin A Dynabeads M-450
(4x108 particles per milliliter). The Dynabeads were
allowed to adhere to the erythrocytes for 30 minutes at 4°C with
continuous shaking before they were removed by means of a magnet. The
erythrocyte-free mononuclear cell fraction was counted in a Coulter Z1
counter, centrifuged (740g for 10 minutes), and
resuspended in Ham's F-10 with gentamicin (250 µg/mL).
Oxidation of LDL
Cell-mediated oxidation of LDL was performed on freshly isolated
LDL at baseline and after 6 weeks of supplementation.
Cu2+-catalyzed and AAPH-initiated oxidations were carried
out on LDL that had been stored in 10% sucrose at -80°C. LDL
isolated at baseline and after supplementation was thawed and dialyzed
simultaneously and immediately subjected to Cu2
+- or AAPH-initiated oxidation. Thawed LDL samples were stored
at 4°C under N2.
Cu2+-Induced Oxidation of LDL
LDL (100 µg/mL) was incubated at 37°C for 0, 1, or 3
hours in the presence of freshly prepared 5 µmol/L
CuSO4 (final concentration). The oxidation was stopped on
ice with the immediate addition of EDTA and BHT (200
µmol/L and 40 µmol/L, final concentrations,
respectively). The intra-assay coefficient of variation for
Cu2+-catalyzed oxidized LDL (n=8) was 4.6% when measured
as the amount of lipid peroxides formed and 1% when measured as
changes in REM.
AAPH-Initiated Oxidation of LDL
LDL was subjected to oxidation initiated by AAPH, a
water-soluble azo compound that thermally decomposes to produce peroxyl
radicals at a constant rate within the water phase.29 30
The oxidation of LDL (100 µg/mL) was carried out at 37°C for
0, 2, or 4 hours in the presence of 4 mmol/L AAPH dissolved
in PBS. The oxidation was stopped on ice with the immediate addition of
EDTA and BHT as described above. The intra-assay coefficient of
variation of AAPH-induced oxidation of LDL (n=8) was 6% when measured
as amount of lipid peroxides formed and 10% when measured as
changes in REM.
Cell-Mediated Oxidation of LDL
Immediately after dialysis, the freshly isolated LDL was
subjected to cell-mediated oxidation by the patients' own
peripheral blood mononuclear cells. The freshly isolated
cells were seeded in triplicate in 24-well tissue-culture plates at a
density of 2x106 mL-1. All
experiments were performed in Ham's F-10 with gentamicin (250
µg/mL) in a total volume of 250 µL per well. The oxidation
was carried out for 0, 3, or 6 hours at 37°C (in a 95% air and 5%
CO2 atmosphere) in the presence of LDL (100
µg/mL), PMA (100 ng/mL), and Cu2+ (2.5
µmol/L). The oxidation was stopped on ice with the immediate
addition of EDTA and BHT. Microscopic examination of the cells was
performed at the end of the incubation.
Lag Time, Formation Rate, and Amount of CDs
The kinetics of Cu2+- and AAPH-induced oxidation of
LDL was followed by determining the changes in absorbance at 234
nm31 using a Beckman DU 640 spectrophotometer with a
12-position micromulticell. LDL (25 µg/mL) was incubated at
37°C in the presence of 5 µmol/L CuSO4 or
1 mmol/L AAPH. The absorbance was measured every 5 minutes
for 240 minutes. When AAPH was used to induce oxidation, AAPH dissolved
in PBS was used as a blank, and the absorbance values of the samples
were corrected according to changes in the absorbance of the blank.
From these analyses, the lag time (in minutes) was calculated
as the interval between t=0 and the intercept of the linear slope of
the curve with the x axis. The formation rate of CDs
(nmol · mg LDL protein-1 ·
min-1) and the maximum amount of CDs formed
(nmol/mg LDL protein) were calculated using a molar extinction
coefficient of E234 nm=2.52x104 L ·
mol-1 ·
cm-1.32 The intra-assay
coefficients of variation were 9.4%, 5.4%, and 1.1% for lag time,
formation rate, and maximum amount of CDs formed, respectively, for
Cu2+-catalyzed oxidation (n=12) and 4.8%, 6.5%, and 4.5%
for lag time, formation rate, and maximum amount of CDs formed,
respectively, for AAPH-induced oxidation (n=10).
Lipid Peroxides in Oxidized LDL
The amount of lipid peroxides in Cu2+-catalyzed,
AAPH-induced, and cell-mediated oxidized LDL was determined by a
colorimetric end point kit in which hemoglobin
catalyzes the reaction of hydroperoxides with a methylene blue
derivative, forming an equimolar concentration of methylene
blue.33 The amount of lipid peroxides was calculated using
cumene hydroperoxide as the standard and is given as nanomoles per
milligram LDL protein. The intra-assay coefficient of variation was
<2% (n=10).
REM of Oxidized LDL
Changes in net negative surface charge of
Cu2+-catalyzed, AAPH-induced, and cell-mediated oxidized
LDL were determined by 0.5% agarose gel electrophoresis (Paragon) in
0.05 mol/L barbital buffer, pH 8.6, after the LDLs were stained
with Sudan black B.34 REM was calculated as the mobility
of oxidized LDL relative to that of native LDL. The intra-assay
coefficient of variation was <1% (n=10).
Surface Charge and Particle Size of Native LDL
The electrophoretic mobility of native LDL particles was
measured on Corning precast 1% agarose gels.35 The
surface potential (S) was calculated from the equation
S=(Ve/Pe)6
n/D, where Ve is the
electrophoretic velocity (migration distance [cm]/time [30
minutes]), Pe is the electrophoretic potential (voltage
applied [100 V]/gel distance [5.5 cm]), n is the coefficient of
viscosity (0.0089 poise), and D is the solvent dielectric constant
(78.36). A detailed justification for the use of this equation is given
by Sparks and Phillips.35 The net number of negative
charges per LDL particle (valence, V) was also
calculated.35 The particle size of native LDL was
determined by negative-staining electron microscopy.36
FA Concentrations in Serum Phospholipids
Serum lipids were extracted in ethanol with C17:0 as the
internal standard.37 The phospholipids were isolated on
small aminopropyl columns (Waters SepPack).38 The FA
concentrations of serum phospholipids were determined by gas-liquid
chromatography of the FA methyl esters on a
fused-silica wall-coated capillary column (Chrompack CP-Wax-52 CB, 25
mx0.25 mm) in a Perkin-Elmer Auto System essentially as described
by Tande et al.39 Identification of major peaks was made
by comparing the retention time with those of standard FA methyl
esters. A test serum was analyzed with every new series of
serum samples to test the reproducibility of the analyses.
FA Concentrations in Native LDL and Mononuclear Cells
The FA concentrations of LDL and mononuclear cells were
determined after extraction with the method of Folch et
al40 by gas-liquid chromatography of the
FA methyl esters as described above with C17:0 as the internal
standard.
Vitamin E Concentration
Vitamin E concentration in serum was measured by a
Shimadzu/Waters high-performance liquid
chromatography system. Serum was extracted with
absolute 2-propanol in an amber vial and centrifuged at 4000
rpm for 15 minutes at 4°C. The vial was loaded into a cooled
autoinjector, and an aliquot (20 µL) of the supernatant was injected
into the system. The analytical column was a Supelcosil LC-8
250x4.6 mm, and the mobile phase consisted of
acetonitrile/methyl-tert-butyl ether/water (1:64:35,
vol/vol/vol). The flow rate was 1.2 mL/min and the
temperature was ambient. Detection was carried out with
fluorescence (excitation, 294 nm; emission, 330 nm). Vitamin E
concentration was determined from the peak height of a five-point
external standard. The calibration curve was linear at least in the
range 0.5 to 40 µmol/L (r2=.997).
The intra- assay coefficient of variation was <3% (n=21). The
recovery of vitamin E was 96.2±5.1% (5 µmol/L),
99.1±4.6% (20 µmol/L), and 101.0±4.1% (40
µmol/L) (n=6 for each concentration).
Routine Laboratory Assays
Venous blood samples for measurement of routine laboratory
analyses were obtained from the subjects before and after
supplementation.
Statistical Methods
Median values together with min/max values are
presented. Nonparametric statistical methods were
chosen because most of the variables were skewed and the number of
observations limited. The Kruskal-Wallis test41 was used
to examine whether there were differences between the four groups. When
a statistically significant effect was observed (P<.05),
pairwise comparisons between groups were performed by Mann-Whitney
U tests. Data analysis was performed using the
statistical package Minitab release 9.
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Results |
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Characteristics of the Patients
Daily Dietary Intake
Daily dietary intake of relevant nutrients was assessed by quantitative food-frequency questionnaires. At baseline there were no significant differences among the groups (Table 2
). The dietary intake of
¯-3 FAs ranged from 0.8 to 6.4 g/d (Table 2),
with a median value of 2.3 g/d, of which EPA and DHA constituted
0.7 g/d (range, 0.06 to 3.4 g/d; n=41), whereas 18:3
-3 constituted 1.5 g/d (range, 0.6 to 4.9 g/d; n=41).
The median value for fish intake was 85 g/d (range, 12 to 358
g/d), of which fatty fish like salmon, trout, mackerel, and
herring comprised 83 g/d (range, 0-134 g/d; n=41).
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Biological Features
There were no significant differences in biological features of
the participants at baseline (Table 3).
Furthermore, no significant changes occurred among the groups during
the supplementation period with respect to body weight, blood pressure,
total cholesterol, HDL cholesterol, LDL
cholesterol, or any of the measured parameters
presented in Table 3. Triacylglycerol levels were reduced in 16
of 22 patients supplemented with ¯-3 FAs (Table 4). These changes were not significantly
different from that of the control group. However,
triacylglycerol levels were significantly lowered
by 25% (n=22) versus 9.6% in the groups not receiving
¯-3 FAs (n=19; P<.03).
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Serum Concentrations of FAs and Vitamin E
Supplementation with ¯-3 FAs or
¯-3 FA/antioxidants resulted in significantly higher
concentration of ¯-3 FAs and lower concentration of
¯-6 FAs in serum phospholipids compared with
antioxidant supplementation alone and control, whereas no significant
changes occurred among groups in the total amount of polyunsaturated
FAs (Table 4
). Supplementation with FA control capsules did not alter
the content of any of the FAs of serum phospholipids.
Vitamin E concentrations in serum at baseline ranged from 31 to 76
µmol/L, with a median value of 46 µmol/L (n=41),
indicating that none of the smokers had lower levels of plasma vitamin
E than the lowest reference value of 14 µmol/L.
Supplementation with the mixture of antioxidants (75 mg vitamin E, 150
mg vitamin C, 15 mg ß-carotene, and 30 mg coenzyme Q10)
significantly changed the serum concentration of vitamin E among the
groups (Table 4). Thus, vitamin E concentration was significantly
higher in the antioxidant group compared with the ¯-3FA
and control groups, whereas vitamin E concentration was significantly
lower in the ¯-3FA group compared with the antioxidant
and control groups. The concentration of vitamin E in the
¯-3FA/antioxidant group was not significantly different
from those of the three other groups.
Consistent with these findings, the lipid-adjusted
vitamin E concentration was significantly higher in the antioxidant
group compared with the control group, whereas the vitamin
E–to–cholesterol plus triacylglycerol
ratio was significantly lower in the ¯-3FA group
compared with the antioxidant, ¯-3FA/antioxidant, and
control groups (Table 4). Increased concentrations of
¯-3FAs in serum phospholipids from patients
supplemented with ¯-3FAs (Table 4) and increased
vitamin E concentrations measured in sera from patients receiving
antioxidants (Table 4) suggest some degree of compliance.
Characteristics of Native LDL
Lipid Composition
No significant changes were observed among groups in the particle
composition of LDL with respect to cholesterol,
phospholipids, triacylglycerol, and protein (Table 5). As for serum phospholipids,
supplementation with ¯-3FAs alone or
-3FA/antioxidants resulted in significantly higher concentrations of
¯-3FAs in LDL compared with the antioxidant
supplementation and control groups, whereas no significant changes
occurred among groups in the concentrations of ¯-6 or
total polyunsaturated FAs (Table 5). The unsaturation index (a measure
of the FA concentrations as well as the number of double bonds) was
significantly higher in LDL after supplementation with
¯-3FAs compared with control and antioxidant LDL groups
(Table 5).
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LDL Surface Charge and Particle Size
LDL surface charge and particle size were measured to determine
whether any of the treatments might alter (1) the physical state of the
core lipids and thereby, apo B conformation and/or (2) the particle
size and consequently, apo B conformation. Changes in apo B
conformation may lead to an altered charge distribution, and
consequently interferes with LDL receptor–mediated uptake, since it
has been shown that the interaction between LDL particles and the LDL
receptors is electrostatic. In addition, it might lead to changes in
the susceptibility of LDL to oxidative modification, since it has been
shown that LDL subpopulations differ markedly in their capacity to
resist oxidative stress in vitro.
The results showed that supplementation with
¯-3FAs, moderate amounts of antioxidants, or a mixture
of the two resulted in no significant differences between groups in the
surface potential and valence of LDL particles (Table 5). The median
particle size of LDL at baseline was 265 Å (258 and 267 Å, min/max
values, respectively; n=34, data not shown). None of the
supplementation regimens significantly changed LDL particle size. Since
no differences among groups in terms of LDL particle composition, size,
and surface charge were observed, these findings suggest that there
were no significant changes in apo B-100 conformation by the various
treatments.
Oxidation of LDL
Cu2+-Catalyzed Oxidation
No significant changes occurred in the oxidizability
parameters among LDLs isolated from subjects supplied with
¯-3FAs, ¯-3FAs/antioxidant, or control
FAs (Figs 1 and 2),
except that the REM of
¯-3FA–and ¯-3FA/antioxidant–enriched
LDL was significantly higher than that of antioxidant and control LDL
after a 1-hour oxidation (Table 6).
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Antioxidant-enriched LDL showed significantly higher resistance to
oxidation than LDL from the other three treatment groups. Thus, the lag
time for formation of CDs was significantly longer compared with that
in the other groups (Fig 1). Furthermore, the amount of lipid peroxides
formed in antioxidant-enriched LDL during the early phase of oxidation
(1 hour) was significantly lower compared with that of
¯-3FA–enriched LDL and control LDL (Fig 2).
Antioxidants affected neither the rate of oxidation nor the amounts of
CDs and lipid peroxides formed later in the peroxidation cascade.
However, antioxidant-enriched LDL showed significantly lower REM than
LDL from the three other supplementation groups (Table 6).
AAPH-Induced Oxidation
The oxidation rate of ¯-3FA–enriched LDL
was significantly slower than that of control-LDL after 6 weeks of
treatment (Fig 3B). Furthermore, the REM
of antioxidant-enriched LDL was lower than that of
¯-3FA–enriched LDL (Table 6). No significant
differences occurred among groups with respect to lag time or amounts
of CDs or lipid peroxides formed (Figs 3 and 4).
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Cell-Mediated Oxidation
Supplementation with ¯-3FAs increased the
concentration of ¯-3FAs in mononuclear cells from 14 of
18 patients (Table 7). Changes observed
in the ¯-3FA groups were not significantly different
from that of the control group. However, ¯-3FA
concentrations significantly increased by 42% (n=18) versus a fall of
11% in the groups not receiving ¯-3FAs (n=16;
P<.05). Simultaneously, ¯-6 FA
concentrations in the groups receiving ¯-3FAs were
significantly reduced by 22% compared with a reduction of 1% in the
groups not receiving ¯-3FAs (P<.04),
whereas no significant changes were measured in cellular concentrations
of polyunsaturated FAs. The cells from the four patients who did not
exhibit increased incorporation of ¯-3FAs showed very
high baseline cellular ¯-3FA contents (2-fold that of
baseline median values), which may explain why they did not increase
further. When LDL was subjected to cell-mediated oxidation, no
significant differences were observed among any of the treatment groups
with respect to the amounts of lipid peroxides generated during
oxidation or changes in the REM of LDL (Table 8).
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Discussion |
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The present study has demonstrated that supplementation with ¯-3FAs alone or in combination with antioxidants to male smokers with combined hyperlipidemia did not render their LDL particles more susceptible to in vitro oxidation. Moreover, supplementation with moderate amounts of antioxidants protected LDL against in vitro oxidation in the early phases of the oxidative process, probably by delaying the onset of oxidation (Table 9
).
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Effects of ¯-3FAs
The present data concerning the effects of fish oil
supplementation on the susceptibility of LDL to oxidation support and
extend our previous findings.12 13 The present study
was a randomized, double-blind, placebo-controlled trial in which
changes between groups were compared. When values after treatment were
compared with baseline values within the ¯-3FA group
alone, some of the measured parameters suggested that
¯-3FAs promoted LDL oxidation, although no significant
differences between the ¯-3FA and control groups were
observed. This notion may underline the importance of
placebo-controlled trials when LDL oxidizability is evaluated. One
limitation with a placebo-controlled study including isolation of LDL
as well as mononuclear cells is the number of participants. It could be
argued that 11 subjects per group are rather few. However, the subjects
were studied extensively by utilizing three different methods to
initiate LDL oxidation as well as by five parameters to
assess the extent of oxidation; all methods had low intra-assay
coefficients of variation. In addition, precautions were taken to
minimize possible interassay variations before and after treatment.
Thus, all LDL samples were stored frozen in the presence of sucrose at
-80°C until the end of the supplementation period. Then the two LDL
samples isolated from the same subject at baseline and after 6 weeks of
supplementation were oxidized by Cu2 + and AAPH and
analyzed further during the same day. With 
set at.05, our
study had 70% to 75% chance of detecting a 5 nmol ·
mg-1 · min-1
change in formation rate, a 200 nmol/mg change in lipid
peroxides (1 hour of oxidation), and a change of 0.5 REM (1 hour of
oxidation) in the ¯-3FA group. Similarly, our study had
an 80% chance of discovering a 10-minute change in lag time in the
antioxidant group.
When copper ions are used to catalyze oxidation, copper binds to the surface of LDL particles and promotes oxidation by catalyzing the breakdown of preformed lipid peroxides, which further generate lipid peroxides in a chain reaction.42 The azo compound AAPH induces oxidation by a constant, temperature-dependent generation of peroxyl radicals in the aqueous phase.43 AAPH does not require binding to LDL for its activity and can act without the presence of preformed lipid peroxides. Since ¯-3FAs may affect cells that are crucial for initiating LDL oxidation in vivo, the patients' own mononuclear cells were used to oxidize autologous LDL in an attempt to better reflect the events occurring in the vessel wall. Peripheral blood mononuclear cells mediate oxidation mainly by NADPH oxidase–derived free radicals.44 Whether copper ion–, peroxyl radical–, or cell-mediated oxidation best reflect the oxidative conditions in vivo is not known. Interestingly, dietary supplementation with ¯-3FAs reduced the release of superoxide anions from human monocytes.23 24 On the other hand, in vitro incorporation of ¯-3FAs in U937 cells enhanced lipid radical formation after iron-induced oxidative stress.45 Likewise, in vitro enrichment of J774 cells with fish oil enhanced the capability of the cells to oxidize LDL.46
¯-3FAs decreased the rate by which AAPH-induced oxidation occurred. A reduced propagation rate combined with increases in other oxidizability parameters have previously been reported in ¯-3FA–supplemented humans11 17 and minipigs.20 Thomas et al19 reported that the propagation rate of ¯-3FA–enriched LDL from cynomolgus monkeys was similar to monounsaturated and saturated FA–enriched LDL, whereas the rate of propagation was significantly higher for ¯-6 FA–enriched LDL particles. One speculation partly explaining the reduced propagation rate with ¯-3FAs could be that the tight packing of EPA and DHA in complex membrane lipids makes the double bounds less available for free-radical interaction.47
Since no significant differences were observed among groups in the
maximum amounts of CDs formed and in lipid peroxides after 3 or 4 hours
of oxidation, this may suggest that the availability of substrate for
oxidative modification was similar in LDL from all treatment groups. A
possible explanation for the fall in ¯-6 FAs observed
concomitant with the increase in ¯-3FAs could be
competition between the two types of polyunsaturated
FAs.48 Since ¯-3FA supplementation
increased the unsaturation index of LDL compared with control while the
relative content of total polyunsaturated FAs in LDL remained
unchanged, these findings may suggest that the concentration of the
polyunsaturates was a more important factor affecting oxidation than
was the degree of unsaturation. These findings are consistent
with data from Thomas et al,19 showing that
substituting 
40% of the linoleic acid with more unsaturated FAs did
not alter the propagation rate. In other words, so long as the
concentration of ¯-3FAs increases at the expense of
¯-6 FAs, the susceptibility of LDL to oxidation may be
unchanged, despite an increased number of double bonds (Table 9).
In a previous study we observed significantly enhanced susceptibility of LDL to Cu2+-catalyzed oxidation after 3 weeks of fish oil supplementation compared with baseline values, whereas after 6 weeks no significant differences were observed compared with baseline values or controls.13 These results may suggest a possible modulating effect of ¯-3FAs on endogenous antioxidant systems. This notion is supported by the finding that oral administration of EPA enhanced the hepatic antioxidant defense in mice.49 Such an adaptive effect could provide an explanation as to why enhanced LDL oxidizability was not observed after ¯-3FA supplementation, despite lower vitamin E concentrations. In addition, a possible adaptation may explain some of the inconsistent results reported on fish oil effects on LDL oxidation.7 8 9 10 11 12 13 14 15 16 17 Thus, enhanced oxidizability should be seen more often after short-term compared with long-term supplementation periods. In line with this, the supplementation periods were 3, 4, or 6 weeks in the studies suggesting increased LDL oxidizability,7 8 9 10 11 whereas they were 5, 6, 8, 12, or 16 weeks in the studies suggesting no effect.12 13 14 15 16 17
In addition to differences among studies in duration of supplementation period, type of patients included, and amount of ¯-3FAs provided,7 8 9 10 11 12 13 14 15 16 17 different experimental conditions may explain some of the apparently conflicting results obtained regarding fish oil effects on LDL oxidation. In some of the studies conclusions were based partly7 9 11 14 or solely8 on the amount of TBARS formed during oxidation. Because TBARS are generated as oxidation products of FAs containing three or more double bonds, variations in TBARS production reflect the lipid composition rather than the susceptibility to lipid peroxidation.32
Triacylglycerol levels in patients supplemented
with ¯-3FAs were lowered by 25%. This change was not
significantly different from the change in the control group. However,
the percent change was significantly different from that of the groups
not receiving ¯-3FAs. Failure of ¯-3FAs
to significantly lower triacylglycerol levels in
normolipidemic individuals occurred in about half of the
placebo-controlled studies reported.50 Similar lack of a
significant reduction in triacylglycerol have also
been reported in hypertriglyceridemic
patients after 4 weeks of supplementation with EPA and DHA (5
g/d).51 One possible explanation for the lack of a
significant lowering effect of ¯-3FAs might be that the
serum concentration of triacylglycerols exhibits
large variations, depending on the dietary fat intake. The background
intake of EPA and DHA in our group (Table 4) was comparable to that of
male US health professionals 40 to 75 years of age (ranging from 0.01
to 6.5 g/d, with median values of 0.07 and 0.58 g/d in
lower and upper quintiles, respectively52 ).
Effects of Antioxidants
In the present trial, supplementation with moderate amounts of
vitamin E, vitamin C, ß-carotene, and coenzyme Q10
protected LDL against oxidation when incubated with copper ions.
Increased resistance to and reduced oxidizability to
Cu2+-induced oxidation after supplementation with
antioxidant "cocktails" have previously been reported by several
authors.53 54 55 56 57 58 However, except for the study by Mackness et
al,55 antioxidants were supplied in much higher doses than
those used in the current study. Thus, -tocopherol was
given in dosages from 200 to 1600 mg/d compared with 75 mg in
our study. Moreover, the doses of vitamin C varied from 400 to 2000
mg/d compared with 150 mg in the present study. Some of the
studies53 56 showed that combined supplementations were
not superior to high dose of -tocopherol alone. Other
studies have shown that supplementation with vitamin E
alone53 59 60 61 62 63 64 but not with ß-carotene
alone53 62 65 protects LDL against oxidation in vitro.
These data are consistent with findings in four recently
reported trials with clinical end points.66 67 68 69 Thus, an
inverse relationship was observed between intake of vitamin E and risk
of developing angina pectoris among male smokers66 and
that of nonfatal myocardial infarction among patients with
angiographically proven coronary
atherosclerosis.69 Interestingly, the dose
of vitamin E used in the primary prevention study was as low as 50
mg/d.66 In contrast, ß-carotene supplementation
to male physicians produced neither benefit nor harm in terms of
incidence of cardiovascular diseases,67
whereas ß-carotene without or with vitamin A given to
smokers66 68 and to workers exposed to
asbestos68 was associated with a slight increase in the
incidence of angina66 and death from
cardiovascular diseases.68
Concluding Remarks
The present randomized, double-blind, placebo-controlled trial
indicates that supplementation with ¯-3FAs alone or in
combination with antioxidants to male smokers with combined
hyperlipidemia did not render LDL particles more
susceptible to in vitro oxidation. Furthermore, supplementation with
moderate amounts of antioxidants alone protected LDL against in vitro
oxidation. The findings regarding effects of ¯-3FAs are
consistent with conclusions drawn in some
studies12 13 14 15 16 17 but are in conflict with the increased LDL
oxidizability reported in other studies.7 8 9 10 11 In contrast,
studies have consistently shown that linoleic acid–enriched
LDL is more susceptible to oxidation than LDL enriched with oleic
acid.70 71 72 73 74 75 76
Regardless of the effects on LDL oxidizability, antiatherogenic effects of ¯-3FAs have been observed in animal models, including pigs and monkeys.77 78 79 80 In humans, Eritsland et al81 reported that dietary supplementation of ¯-3FAs to patients undergoing coronary artery bypass grafting reduced the incidence of vein graft occlusion. Also, the antiatherogenic potential of ¯-3FAs have been underlined in a prospective intervention study in which a significant reduction in overall mortality was observed in a group of men advised to eat two meals of oily fish per week.82
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by grants from Petter Möller Orkla ASA, The Johan Throne Holst Fund, Freia Chocolade fabriks medisinske Fond, The Norwegian Council on Cardiovascular Disease, and The Eckbo's Fund to the project group. We thank Kjersti Viken, Anne Randi Alvestad, Eva Grabner, Asbjørg Flo, Christer Tungard, and Thomas Gundersen for expert technical assistance.
Received February 4, 1997; accepted June 23, 1997.
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References |
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  K. B. Holven, J. K. Damas, A. Yndestad, T. Waehre, T. Ueland, B. Halvorsen, L. Heggelund, W. J. Sandberg, A. G. Semb, S. S. Froland, et al. Chemokines in Children With Heterozygous Familiar Hypercholesterolemia: Selective Upregulation of RANTES Arterioscler Thromb Vasc Biol, January 1, 2006; 26(1): 200 - 205. [Abstract] [Full Text] [PDF]
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 K. B. Holven, A. M. Myhre, P. Aukrust, T. A. Hagve, L. Ose, and M. S. Nenseter Patients with familial hypercholesterolaemia show enhanced spontaneous chemokine release from peripheral blood mononuclear cells ex vivo: Dependency of xanthomas/xanthelasms, smoking and gender Eur. Heart J., October 1, 2003; 24(19): 1756 - 1762. [Abstract] [Full Text] [PDF]
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 Y. V. Yuan and D. D. Kitts Dietary (n-3) Fat and Cholesterol Alter Tissue Antioxidant Enzymes and Susceptibility to Oxidation in SHR and WKY Rats J. Nutr., March 1, 2003; 133(3): 679 - 688. [Abstract] [Full Text] [PDF]
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P. M. Kris-Etherton, W. S. Harris, L. J. Appel, and for the Nutrition Committee Fish Consumption, Fish Oil, Omega-3 Fatty Acids, and Cardiovascular Disease Arterioscler Thromb Vasc Biol, February 1, 2003; 23(2): e20 - 30. [Full Text] [PDF]
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 P. M. Kris-Etherton, W. S. Harris, L. J. Appel, and for the Nutrition Committee Fish Consumption, Fish Oil, Omega-3 Fatty Acids, and Cardiovascular Disease Circulation, November 19, 2002; 106(21): 2747 - 2757. [Full Text] [PDF]
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K. B. Holven, P. Aukrust, T. Holm, L. Ose, and M. S. Nenseter Folic Acid Treatment Reduces Chemokine Release From Peripheral Blood Mononuclear Cells in Hyperhomocysteinemic Subjects Arterioscler Thromb Vasc Biol, April 1, 2002; 22(4): 699 - 703. [Abstract] [Full Text] [PDF]
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R. B. Weinberg, B. S. VanderWerken, R. A. Anderson, J. E. Stegner, and M. J. Thomas Pro-Oxidant Effect of Vitamin E in Cigarette Smokers Consuming a High Polyunsaturated Fat Diet Arterioscler Thromb Vasc Biol, June 1, 2001; 21(6): 1029 - 1033. [Abstract] [Full Text] [PDF]
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 J. E. Reseland, F. Haugen, K. Hollung, K. Solvoll, B. Halvorsen, I. R. Brude, M. S. Nenseter, E. N. Christiansen, and C. A. Drevon Reduction of leptin gene expression by dietary polyunsaturated fatty acids J. Lipid Res., May 1, 2001; 42(5): 743 - 750. [Abstract] [Full Text]
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J. V. Higdon, S. H. Du, Y. S. Lee, T. Wu, and R. C. Wander Supplementation of postmenopausal women with fish oil does not increase overall oxidation of LDL ex vivo compared to dietary oils rich in oleate and linoleate J. Lipid Res., March 1, 2001; 42(3): 407 - 418. [Abstract] [Full Text]
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O. Johansen, I. Seljeflot, A. T. Hostmark, and H. Arnesen The Effect of Supplementation With Omega-3 Fatty Acids on Soluble Markers of Endothelial Function in Patients With Coronary Heart Disease Arterioscler Thromb Vasc Biol, July 1, 1999; 19(7): 1681 - 1686. [Abstract] [Full Text] [PDF]
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K. B. Holven, P. Aukrust, T. Holm, L. Ose, and M. S. Nenseter Folic Acid Treatment Reduces Chemokine Release From Peripheral Blood Mononuclear Cells in Hyperhomocysteinemic Subjects Arterioscler Thromb Vasc Biol, April 1, 2002; 22(4): 699 - 703. [Abstract] [Full Text] [PDF]
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